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Wildly popular food cart chain The Halal Guys is now offering the ingredients to its creamy, seasoned white and hot sauces doused over platters of lamb, chicken and rice. The packets contain soybean, canola oil, egg yolk, vinegar, water, salt, sugar, natural flavors, black pepper and common condiment additives like disodium EDTA, xanthan gum and sodium benzoate, DNAinfo reports.
The first three white sauce ingredients comprise the basics used to make mayonnaise, while the a€?natural flavorsa€? are kept under wraps.
The fiery hot sauce includes ground red pepper, vinegar, salt, spices and concentrated lemon juice. This year, the street food concept opened its first brick-and-mortar location in June on E. Science, Technology and Medicine open access publisher.Publish, read and share novel research. Application of Amylolytic Lactobacillus fermentum 04BBA19 in Fermentation for Simultaneous Production of Thermostable ?-Amylase and Lactic AcidBertrand Tatsinkou Fossi1 and Frederic Tavea2[1] Department of Microbiology and Parasitology, University of Buea, Buea, Cameroon[2] Department of Biochemistry, University of Douala, Douala, Cameroon1.
Yams, red skinned potato and white potatoes fresh onions, parsley tossed in a creamy dressing.
Yams, potatoes, dressing (water, vegetable oil, sugar, vinegar, modified corn starch, liquid egg yolk, salt,lemon juice concentrate, calcium disodium EDTA), onions, parsley, salt, pepper. Plate assays for detection of amylase activity of lactic acid bacteria (04BBA19, 26BMB81) on MRS-starch agar plate medium. IntroductionLactic acid bacteria (LAB) have diverse applications for both animals and humans. The diameter of hydrolysis halo was revealed by flooding the plates with Iodine solution (0.1% I2+1% KI) after 48 h of culture at 40°CTable 1. Food, pharmaceutical and chemical industries rely on these microorganisms to produce fermented beverage, foods and other important compounds of industrial interests.
The enzyme was pre-incubated at optimum pH, for 30, 60, 90, 120 and 180 min at temperatures (80, 90 and 100°C).
In recent years the industrial relevance of lactic acid bacteria is on an increasing trend because of the application of lactic acid as chemical for the production of biodegradable plastics [1]. The remaining activity was determined incubating the enzyme at optimum temperature, 60°C for 30 min.
Typical LAB are Gram-positive, non-sporing, catalase-negative, devoid of cytochromes, anaerobic but aerotolerant cocci or rods that are acid-tolerant and produce lactic acid as the major end product during sugar fermentation [2]. Although most LAB are unable to degrade starch because of the lack of the amylolytic activity, a few exhibit this activity and are qualified as amylolytic lactic acid bacteria (ALAB) which are able to decompose starchy material through the amylases production during the fermentation processes [3].
Regarding the importance and availability of starchy biomass in the world, amylases and lactic acid production from starch appear as two potential industrial applications of ALAB. Amylases play important role in degradation of starch and are produced in bulk from microorganisms and represent about 25 to 33% of the world enzyme market [4]. The spectrum of amylases application has widened in many fields, such as clinical, medical and analytical chemistry as well as in the textile, food, fermentation, paper, distillery and brewing industries [4].
The advantages of using thermostable amylases in industrial processes include the decreased risk of contamination, cost of external cooling and increased diffusion rate [4]. The use of thermostable amylases from Lactobacillus is of advantage as they are generally non-pathogenic.
On the other hand, the major end product of LAB fermentation, lactate, has applications as a preservative, acidulant and flavouring agent in the food industry, because of the tartness provided by lactate and also because lactate is generally regarded as safe (GRAS) [6].Thus a thermostable amylase producing lactic acid bacterium would be a potential candidate for food industries and especially for the making of high density gruel from starchy raw material as corn or wheat [7]. This would require a good knowledge of the conditions required to optimally produce amylase and lactic acid of good quality.
The present study deals with the co-production of thermostable ?-amylase and lactic acid from a LAB, Lactobacillus fermentum 04BBA19, isolated from a starchy waste of a soil sample from the western region of Cameroon.2. Amylolytic lactic acid bacteriaAmylolytic lactic acid bacteria (ALAB) have been reported from different tropical starchy fermented foods, made especially from roots as cassava and sweet potato or grains as maize sorghum and rice. Strains of Lactobacillus plantarum have been isolated from African cassava-based fermented products [8], L. Amylolytic strains of Lactobacillus fermentum were isolated for the first time from Benin maize sourdough (ogi and mawe) by Agati et al. Owing to their relatively high starch content, starchy biomass appears as an important eco-niche for the screening and isolation of ALAB, which can be industrially applied to convert starch into mono- and disaccharides for lactic acid fermentation. The composition of the microbiota and in particular the occurrence of ALAB is determined by the way the raw material is processed [13]. Most ALAB isolated belong to the Lactobacillus genus, however few studies reported the existence of amylolytic activity in some strains of Bifidobacterium isolated from the human large intestinal tract [14, 15].


The distribution of amylolytic microorganisms in the human large intestinal tract has been investigated in various individuals of different ages using anaerobic cultures techniques.
So far, twenty one amylolytic bifidobacteria have been isolated from adult faeces and tested for rice fermentation [16]. Owing to the ability of their ?-amylases to partially hydrolyze raw starch, ALAB can ferment different types of amylaceous raw material, such as corn [17], potato [18], or cassava [19] and different starchy substrates [20, 21, 8]. Amylolytic LAB utilize starchy biomass and convert it into lactic acid in a single step fermentation. ALAB are mainly used in food fermentation, they are involved in cereal based fermented foods such as European sour rye bread, Asian salt bread, sour porridges, dumplings and non-alcoholic beverage production.
Few of them are used for production of lactic acid in single step fermentation of starch [1].
The common method to produce lactic acid from starchy biomass involves the pretreatment for gelatinisation and hydrolysis (liquefaction and saccharification). The liquefaction of the starch is carried out at high temperatures of 90–130 °C for 15 min followed by enzymatic saccharification to glucose and subsequent conversion of glucose to lactic acid by fermentation [22, 1]. This two-step process involving consecutive enzymatic hydrolysis and fermentation makes it economically unattractive. The bioconversion of carbohydrate materials to lactic acid can be made much more effective by coupling the enzymatic hydrolysis of carbohydrate substrates and microbial fermentation of the derived glucose into a single step. This has been successfully employed for lactic acid production from raw starch materials with many representative bacteria including Lactobacillus and Lactococcus species [23, 20, 24, 21].Because at industrial scale, the use of glucose addition is an expensive alternative, there is interest in the use of a cheaper source of carbon, such as starch, the most abundantly available raw material on earth next to cellulose. This, in combination with amylolytic lactic acid bacteria may help to decrease the cost of the overall fermentation process. Development of production strains which ferment starch to lactic acid in a single step is necessary to make the process economical. Very few bacteria have been reported so far for direct fermentation of starch to lactic acid [1, 25, 26] Approximately 3.5 billion tonnes of agricultural residues are produced per annum in the world [27]. The use of a specific carbohydrate feedstock depends on its price, availability, and purity.
Although agro-industrial residues are rich in carbohydrates, their utilization is limited [27].
Sucrose-containing materials such as molasses are commonly exploited raw materials for lactic acid production.
Starch produced from various plant products is a potentially interesting raw material based on cost and availability. Laboratory-scale fermentations have been reported for lactic acid production from starch by Lactobacillus amylophilus GV6 [20], L. Thermostable amylasesAmylases are among the most important enzymes and are of great significance in present-day biotechnology. Although they can be derived from several sources, such as plants, animals and microorganisms; enzymes from microbial sources generally meet industrial demands. The spectrum of amylase application has widened in many other fields, such as clinical, medical and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, food, brewing and distilling industries.
Thermostability is one of the main features of many enzymes sold for bulk industrial usage. Thermostable ?-amylases are of interest because of their potential industrial applications.
They have extensive commercial applications in starch liquefaction, brewing, sizing in textile industries, paper and detergent manufacturing processes. The advantages of using thermostable amylases in industrial processes include the decreased risk of contamination and cost of external cooling, a better solubility of substrates, a lower viscosity allowing accelerated mixing and pumping [36]. Lactobacillus amylovorus, Lactobacillus plantarum, Lactobacillus manihotivorans, and Lactobacillus fermentum are some of the lactic acid bacteria exhibiting amylolytic activity which have been studied [37, 10, 38, 5, 39, 40].
However, most of ?-amylase from these bacteria presented weak thermostability compared to those of genus Bacillus. Owing to the important acidification of fermenting medium by most lactic acid bacteria, the production of thermostable amylase by a lactic acid bacterium under submerged or solid-state fermentation can help to reduce the risk of contamination caused by undesirable micro-organisms during the process [41, 42]. Lactic acidLactic acid a water soluble and highly hygroscopic aliphatic acid is present in humans, animals and microorganisms. It is the first biotechnologically produced multi-functional versatile organic acid having wide range of applications [1], namely as a preservative in many food products. It is non-volatile, odorless organic acid and is classified as GRAS (Generally Recognized As Safe) for use as a general purpose food additive. The lactic acid consumption market is dominated by the food and beverage sector since 1982 [1]. More than 50% of lactic acid produced is used as emulsifying agent in bakery products [44].
Lactic acid or its salts are used in the disinfection and packaging of carcasses, particularly those of poultry and fish, where the addition of aqueous solutions during processing increased shelf life and reduced microbial spoilage.
The esters of calcium and sodium salts of lactate with longer chain fatty acids have been used as very good dough conditioners and emulsifiers in bakery products. The water retaining capacity of lactic acid makes it suitable for use as moisturizer in cosmetic formulations. The natural occurrence of lactic acid in human body makes it very useful as an active ingredient in cosmetics [45].


Lactic acid has long been used in pharmaceutical formulations, mainly in topical ointments, lotions, and parenteral solutions.
It also finds applications in the preparation of biodegradable polymers for medical uses such as surgical sutures, prostheses and controlled drug delivery systems [45]. Because of ever-increasing amount of plastic wastes worldwide, considerable research and development efforts have been devoted towards making a single-use, biodegradable substitute of conventional thermoplastics. Biodegradable polymers are classified as a family of polymers that will degrade completely – either into the corresponding monomers or into products, which are otherwise part of nature – through metabolic action of living organisms.
The demand for lactic acid has been increasing considerably, owing to the promising applications of its polymer, the polylactic acid (PLA), as an environment-friendly alternative to plastics derived from petrochemicals. PLA has received considerable attention as the precursor for the synthesis of biodegradable plastic [46]. The lactic acid polymers have potentially large markets, as they many advantage like biodegradability, thermo plasticity, high strength etc., have potentially large markets.
The substitution of existing synthetic polymers by biodegradable ones would also significantly alleviate waste disposal problems. As the physical properties of PLA depend on the isomeric composition of lactic acid, the production of optically pure lactic acid is essential for polymerization. SamplesTwenty-eight samples of soils were collected from main geographic zones of Cameroon in four localities: (Ngaoundere, Yaounde, Bafoussam and Mbouda) at the factories where starchy wastes are frequently submitted to natural fermentation. Four kinds of factories were investigated: “gari” factories, corn and cassava mills, cassava plantation after harvesting and treatment of tubers and flour markets. Screening of thermostable amylases and lactic acid producing bacteriaThe starch degrading amylolytic lactic acid bacterial strains were isolated from different samples of soil. Enrichment of thermostable amylases producing bacteria was carried out by heating Erlenmeyer flasks at 90°C for 5 min followed by incubation in an alternative shaker at 37-40°C and speed of 150 oscillations per minute for 24 h. The colonies with the largest halo forming zone were pre-selected and tested for Gram staining and catalase activity.Preliminary tests were carried out to determine the heat stability of the amylase of each isolate as we described previously [47, 48]. The gas production from glucose, growth at different temperature (10, 40, 45 °C) as well as the ability to grow in different concentration of NaCl was determined as described by Schillinger and Lucke [49] and Dykes et al [50]. The isolates which were Gram positive and catalase negative, non-motile and producing heat stable amylase and lactic acid were finally selected and identified using API 50 CH test kit (bioMerieux, France). Optimisation of raw starch degrading thermostable amylase and lactic acid productionThe amylase and lactic acid production was optimized by studying the effect of cultural and environmental variables (carbohydrate and nitrogen sources, metal salts and surfactants) individually and simultaneously.
The suspension was retained for 1 h at 4 °C, and centrifuged at 8000 g for 30 min at the same temperature.
The remaining ?-amylase activity was measured and expressed as the percentage of the activity of untreated control taken as 100%. The effect of metal salts and chelating agent on amylase activity were evaluated by pre-incubating the enzyme in the presence of effectors for 30 min at 60°C.
Analytical methodsCell growth was evaluated by reading the absorbance of culture medium at 600 nm using a Secoman spectrophotometer and numeration of total colony forming unit by 10-fold serial dilution of fermented broth and pour plating on MRS-starch agar (De Man Rogosa and Sharpe medium in which glucose has been replaced by soluble starch (Prolabo-Merck Eurolab, France)).
In order to evaluate the capacity of microorganism to acidify the culture medium, the pH of the fermented broth was measured using an electronic pH meter (Mettler Seven S20, Japan) The amylolytic power of Lactic acid bacteria was determined using the method of wells by inoculation of 10 ?l of microbial strain in 4 mm depth micro-wells on the surface of MRS-starch agar plate. The amylolytic power was defined as the average diameter (mm) of hydrolysis halo provoked by a strain after its inoculation in micro-well on MRS-starch agar plate for 48 h incubation at optimum temperature of growth for three assaysThe activity of amylase both in crude and purified extracts was assayed by iodine method. After 30 min the enzyme reaction was stopped by rapidly adding 1ml of 1M HCl into the reaction mixture. For the determination of residual starch, 1 ml of the reaction mixture was added to 2.4 ml of diluted iodine solution and its optical density was read at 620 nm using a spectrophotometer (Secoman). One unit of amylase activity (U) was defined as the amount of enzyme able to hydrolyse 1 g of soluble starch during 60 min under the experimental condition.
The nature of amylase (endo-acting or exo-acting) was determined according to Ceralpha method (Megazyme) which uses a blocked maltoheptaoside as substratre [57]. The affinity of the enzyme preparation from selected LAB toward raw cassava starch was studied by incubating 0.2 g of raw cassava flour with 1ml of the enzyme solution at 60 ?C for 15 min. After centrifugation, the ?-amylase activity of the supernatant was measured and the adsorption percentage was calculated as follows: Adsorption (%)=A?BA?100, A is the original ?-amylase activity and B is the ?-amylase activity in the supernatant after adsorption on raw potato starch granules.
For the determination of raw starch digestibility, raw cassava was used and the reaction mixture containing 100 U of ?-amylase preparation from the selected LAB and 100 mg of raw cassava starch in a final volume of 10ml dispensed in 100ml Erlenmeyer flasks were incubated in alternative water bath shaker at 60?C and 150 oscillations per min.
After a time interval of 6 h, the reducing sugars liberated in the reaction mixtures were determined by dinitrosalicyclic acid method [58].Light microscopy was used for the examination of the effect of enzyme on raw starch granules using Olympus microscope BH-2.
The amylolytic power is an expression of the capacity of an isolate to degrade starch during the culture. Among the amylase overproducing isolates, two (04BBA19, 26BMB81) were aero-anaerobic non spore forming, gram positive and catalase negative bacteria; this characteristic is proper to lactic acid bacteria. Biochemical characteristics of these isolates were carried out using API 50 CH kit bioMerieux system, the results are summarized in Table 1, the isolates were tested for their possibility to ferment 50 carbohydrates, and this fermentation profile was use for their numerical identification.
According to their biochemical profile, 04BBA19 and 26BMB81 were respectively identified as Lactobacillus fermentum and Lactobacillus plantarum.



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